Soluble expression of humanized anti-cd20 single chain antibody in escherichia coli by cytoplasmic chaperones co-expression

Background: CD20 is an important cell surface receptor that is used for target therapy of B cell lymphoma and some related blood diseases due to vital function of CD20. In previous studies, a Rituximab based humanized single chain variable fragment [scFv] antibody showed good reactivity against B cell related cancer cells. But this recombinant protein produced Inclusion Bodies [IBs] in Escherichia coli [E. coli] cytoplasm .The aim of this study was to investigate the effect of coexpression with cytoplasmic chaperones on expression and solubility of humanized anti-CD20 scFv in E. coli

Methods: For this purpose, the fragment coding for anti-CD20 huscFv subcloned into the pET22b [+] and transformed into the E. coli BL21 [DE3] was evaluated. In order to inhibit the production of IBs, the effects of co-expression with cytoplasmic chaperones GroEL, DnaK, GroES, Tig, DnaJ and GrpE were investigated

Result: Coexpression with cytoplasmic chaperones led to increased soluble expression of anti-CD20 recombinant protein. Among investigated chaperones, pKJE7 chaperone plasmid containing DnaJ, GrpE, DnaK chaperone genes had significant effects with an expression yield of 325Mug/ml soluble anti-CD20 scFv

Conclusion: The result of this study demonstrated remarkable effect of pKJE7 chaperone on enhancement of soluble expression of anti-CD20 huscFv antibody in E. coli

[Cassette chromosome mec typing of methicillin-resistant staphylococcus aureus isolates collected from Sina and Imam Reza Hospitals of Tabriz]

Backgrounds and Objectives: Methicillin-resistant Staphylococcus aureus is a cause of nosocomial infections leading to high mortality. Since these strains have become prevalent in the world, it is necessary to identify and type them

Materials and Methods: This cross-sectional study was conducted to study a total of 1475 specimens collected from patients of Imam Reza and Sina hospitals of Tabriz in 2012-2013. Using phenotypic tests such as Gram stain, catalase, coagulase, DNase and mannitol fermentation 169 isolates of Staphylococcus aureus and by utilizing methicillin-resistance test 100 MRSA isolates were identified. SCCmec typing was performed by multiplex PCR method and the results were analyzed using chi-square tests using SPSS-18 software

Results: Disc agar diffusion test using cefoxitin disc [30 microg] showed methicillin resistance in 59% of our isolates. mecA and femB genes were identified in all of the MRSA isolates using PCR method. Frequency of SCCmec types and sub-types were as follow: SCCmecIII [77%], SCCmecI [5%], SCCmecIVa [1%], SCCmecIVc [1%], mixed isolates SCCmecIVc-III [1%] and Non typeable isolates [15%]. Non typeable isolates recovered in two groups [10% without any band and 5% of multi-bands III-I]. In this study, 82% of isolates were HA-MRSA, 3% were CA-MRSA and 15% were Non-typeable

Conclusion: In our S. aureus isolates, the prevalence of methicillin resistance was 59%. The most frequent SCCmec type was SCCmecIII [77%]. Our results demonstrated the spread of HA-MRSA isolates in the community and propagating CA-MRSA isolates in the studied hospitals

[Epidemiology and patterns of drug resistance in pulmonary tuberculosis in East Azerbayjan]

Background and Objectives: Multidrug-Resistant Tuberculosis [MDR-TB] has been lead to complexity in succesfull treatment of Tuberculosis [TB]. The aim of this study was to determine the pattern of first and second line drug resistance on pulmonary TB pateints and study their demografic and clinical links

Materials and Methods: In a cross-sectional study in 2011-2012, 105 Mycobacterium Tuberculosis [M.TB] pateints were collected randomly from east azerbayjan province of Iran. After full clinical history and physical evaluation, standard proportion method was performed for detection of drug resistance in M.TB patients

Results: Frequency distribution of the M.TB in various parts of the city was significantly different. The total prevalence of resistance to any drug was 8.57% [6.1% in new cases and 33.3% in previously treated cases]. Two [1.9%] patients were MDR-TB and a case was Extensively Drug-Resistant [XDR]. In the multiple logistic regrassion analysis, odds of resistance to one or more TB drug was significantly more in retreatment group than newly diagnosed group [OR=7.7]. Frequency of resistance to Streptomycin [5.7%] was the highest and resistance to Etambutol was the lowest [0.95%]. Sputum [88.3%] and coughing [86.4%] were the most common symptoms

Conclusion: Monitoring of drug resistance status by rapid teqnniques is necessary and crucial on patients with previous history of TB. Unexpected distribution of the disease in different parts of the city indicates the need for otherstudies

Cloning and expression of the variable regions of anti-EGFR monoclonal antibody in E.coli for production of a single chain antibody

Epidermal growth factor receptor [EGFR] overexpression is a characteristic of several malignancies and could be considered as an excellent target for designing specific inhibitors such as anti-EGFR monoclonal antibodies for cancer therapy. Drawbacks exerted by large sizes of full-length antibodies have lead to the development of single chain antibodies, which benefit from having smaller sizes and short circulation half-lives. The aim of this study was cloning, expression and purification of variable regions of anti-EGFR monoclonal antibody in E. coli for production of single chain antibodies. The RNA, extracted from the C225 hybridoma cells, was reverse transcribed into cDNA and used for PCR amplification of genes encoding light and heavy chains from the variable regions. The PCR products were cloned and expressed in E. coli BL21 for production of a single chain antibody. The expressed protein was analyzed by SDS-PAGE and purified by Ni-NTA affinity chromatography. The reactivity of purified C225-scFv with EGFR-expressing A431 tumor cell line was tested by western blotting and enzyme-linked immunosorbent assays. The results indicated that C225-scFv was highly expressed in E. coli and appeared as a protein with a mass of 27 kDa in the sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE] analysis of the induced cell lysate. Reactivity analysis of the purified C225-scFv with A431 tumor cell line by western blotting and enzyme linked immune sorbant assay [ELISA] revealed high binding affinity of the recombinant C225-scFv to the target cells. The results of this study indicated that C225-scFv is capable of binding to EGFR and could be considered as a useful tool for diagnosis and treatment of EGFR-overexpressing tumor cells

Protective properties of nontoxic recombinant exotoxin A [domain I-II] against pseudomonas aeruginosa infection

Antibiotic resistance and the need for long-term treatments especially for chronic infections necessitate the development of a vaccine against Pseudomonas aeruginosa infection. In this study, recombinant exotoxin A [domains I and II], [ExoA I-II] protein was expressed, purified and its immunological characteristics were evaluated in a mouse model. The genomic DNA was extracted from P. aeruginosa strain PAO1. The DNA encoding for domains I and II of exotoxin A was amplified by PCR and cloned into the pET22b expression vector. The construct was then transformed into E. coli BL21 and the protein expression was evaluated by the SDS-PAGE method. The Ni-NTA affinity chromatography was used for recombinant protein purification. Mice were then immunized subcutaneously on day 0, 21, 42 and 72 with exotoxin A [Domains I, II]. Antibody production was evaluated by the ELISA method. The immunized and control group mice were exposed to an approximate 2 x LD50 [7.5 x 10[7] CFU] of clinical strain of mucoid P. aeruginosa. Sequencing of the cloned gene showed that the sequence of ExoA one-two gene was in accordance with ExoA I-II from P. aeruginosa PAO1. SDS-PAGE analysis indicated the expression of recombinant protein with a molecular weight of 45 KDa. Vaccination with ExoA I-II produced a significant amount of specific IgG antibodies in mice. Also immunization of mice with ExoA one-two increased survival times against intra-peritoneal challenge with an approximate 7.5 x 10[7] CFU [2 x LD50] of clinical strain of P. aeruginosa. Results of this study suggested that recombinant ExoA I-II is a highly immunogenic protein which can be used as a new vaccine candidate against P. aeruginosa

Prevalence and antibiotic resistance patterns of diarrheagenic Escherichia coli isolated from adolescents and adults in Hamedan, Western Iran

Pathogenic strains of Escherichia coli are a common cause of acute infectious diarrhea. The aim of this study was to investigate the frequency, virulence markers and antibiotic resistance patterns of diarrheagenic E. coli [DEC] isolated from adolescents and adults in Hamadan, west of Iran. A total of 187 stool samples were collected from adults with acute diarrhea. Stool culture was performed by conventional methods for enteropathogenic bacteria. Virulence factor genes for DEC were detected by polymerase chain reaction. Antimicrobial susceptibility was tested using the disk diffusion method. Among the 187 patients, 40 [21.4%] were positive for DEC. The most frequently identified DEC was enteropathogenic E. coli [47.5%], followed by enteroaggregative [20%], enterotoxigenic [17.5%] and shiga-toxin producing E. coli [15%]. No isolates of enteroinvasive E. coli were detected. All STEC strains were stx[+] / eaeA[-]. Out of the seven ETEC strains, five [71.4%] produced ST, one [14.3%] produced only LT and one [14.3%] of the isolates produced both ST and LT encoded by est and elt genes, respectively. Among the 40 DEC strains 27[67.5%] were multidrug resistant. DEC contribute to the burden of diarrhea in adults in Hamadan. Enteropathogenic E. coli was the most commonly identified DEC strain in the region studied

Cloning, expression and characterization of recombinant exotoxin A-flagellin fusion protein as a new vaccine candidate against pseudomonas aeruginosa infections

Background: infections due to Pseudomonas aeruginosa are among the leading causes of morbidity and mortality in patients who suffer from impaired immune responses and chronic diseases such as cystic fibrosis. At present, aggressive antibiotic therapy is the only choice for management of P. aeruginosa infections, but emergence of highly resistant strains necessitated the development of novel alternative therapeutics including an effective vaccine. Several P. aeruginosa antigens have been tested for vaccine development, including lipopolysaccharide alone, polysaccharides alginate, extracellular proteins, exotoxin A [exo A] and killed whole cell. However, none of them are currently available clinically

Methods: in this research, recombinant exoA-flagellin [fliC] fusion protein as a cocktail antigen was expressed and purified and its antigenic characteristics were evaluated

Results: expression of recombinant fusion protein by E. coli using pET22b vector resulted in production of exoA-fliC fusion protein in high concentration. Based on Western-blotting results, recombinant fusion protein showed a good antigenic interaction with sera from patients with various P. aeruginosa infections

Conclusion: these results suggested that recombinant exoA-fliC fusion protein can be produced in the laboratory, and tested as a candidate vaccine in P. aeruginosa infections. Iran. Biomed. J. 17 [1]: 1-7, 2013

Mediterranean fever gene analysis in the Azeri Turk population with familial mediterranean fever: evidence for new mutations associated with disease

Familial Mediterranean fever [FMF] is an autosomal recessive disorder characterized by recurrent febrile attacks accompanied by serosal and synovial membrane inflammation. FMF is caused by mutations in the MEFV gene and are found usually among Mediterranean populations, Armenians, Turks, Arabs and Jews. The aim of this study was to determine the frequency of MEFV gene mutations among FMF patients in the Azeri Turk population in North-West of Iran. In this descriptive study, 130 FMF patients with Azeri Turk origin were screened for mutations in four exons [2, 3, 5 and10] of MEFV gene. Genomic DNA was extracted from whole blood and entered in ARMS-PCR and PCR-RFLP reactions. When cases were negative in ARMS-PCR and PCR-RFLP, the exons were amplified and subjected to direct sequencing. Our results showed that the most common mutations in this study population was M694V [40.19%] followed by E148Q [17.64%], V726A [13.72%], M680I [12.74%] and M694I [2.94%] mutations. Four new mutations including K618N, K716M, S614F and G136E were identified in our study. The prevalence of five common mutations in our study was highly similar to previous studies analysing the Mediterranean basin populations. Investigation by sequencing also revealed four new variants in the study population. The main genotype- phenotype correlation finding was the presence of M694V mutation in homozygote or compound heterozygote state in the patients with renal manifestations

Hepatitis B virus genotypes in Eastern Azerbaijan, Northwest Iran

The aim of this study was to investigate, for the first time, the genotype of hepatitis B virus [HBV] among hepatitis patients in Eastern Azerbaijan Province, Northwest Iran. A total of 100 HBV-infected patients were enrolled in this study. Among these, 40 samples were positive for both HBsAg and HBeAg, whereas 60 samples were HBsAg positive and HBe Agnegative. Patients' sera were subjected to DNA extraction and the genotype determined by PCR using type-specific primers. The results of genotyping revealed that genotype D was the only identified genotype in both acute and chronic patients in the study region. Analysis of sequencing results showed 98% - 99% homology with the previously reported HBV genotype D sequences. Genotype D was recognized as the predominant HBV genotype in the studied area. There was no significant relationship between genotype D of HBV and different types of infections

Detection of acute childhood meningitis by PCR, culture and agglutination tests in Tabriz, Iran

Meningitis is one of the hazardous and life threatening infections and is associated with mortality and morbidity. The aim of this study was to determine etiological agents of childhood bacterial meningitis. The culture, Gram staining, agglutination and PCR assays were used to examine CSF specimens from 277 patients with presumed bacterial meningitis for the occurrence of 4 most common infectious agents consist of N. meningitis, H. influnsae, S. pneumoniae and S. agalactiae between 2008 and 2009 at different wards of the Children Hospital of Tabriz. The mean age of patients was 35 +/- 2 [Mean +/- SEM] month, [minimum 11 days maximum14 years], of all cases 59.6% male and 40.4% female. Overall the diagnosis was confirmed with a CSF culture in 11/277 [3.97%], by agglutination test in 14/277 [5.05%]. The isolated bacteria included S. pneumoniae 5 cases, H. influnsae 2 cases, N. meningitis 3 cases and P. aeroginusae 1 case. A positive PCR assay allowed us to diagnose bacterial meningitis in 19 patients [6.8%]. In the present study, we found PCR to be a useful and sensitive method for the detection of bacterial DNA in the CSF samples from suspected meningitis patients. Furthermore, to maximize management of meningitis cases, a combination of culture and PCR is necessary

Optimizing refolding condition for recombinant tissue plasminogen activator

Low molecular size additives such as L-arginine and the redox compounds have been used both in the culture medium and in vitro refolding to increase recombinant proteins production. Additives increase protein refolding and yield of active proteins by suppressing aggregate formation or enhancing refolding process. In this work, a comparative study was performed on refolding of recombinant plasminogen activator [rPA] in the presence of different concentrations of denaturants and additives. Escherichia coli-expressed rPA inclusion bodies were solubilized in chaotropic denaturants and subjected to protein refolding by dilution method. The effects of various additives, the impact of pH, residual Guanidin Hydrochloride [Gn-HCl] and Dithiothreitol [DTT] on refolding process were investigated. The refolding process was assessed by determination of protein solubility and biological assay. The results of the study demonstrated that the best condition for solubilizing the rPA inclusion body was 6M guanidine hydrochloride at pH=10.In refolding step, Larginine showed increasing effect on suppression of aggregation at concentrations of 200-1000 mM. Glutathione pairs [GSH-GssG] showed refolding enhancer effect in a range of 2-20 mM. The highest refolding yield was obtained in 500 mM L-arginine and reduced/oxidized glutathione 10:1 ratio in pH 10.In conclusion, the results show that L-arginine plays an important role in the refolding of human PA, preventing the aggregation of folding intermediate, and glutathione pair is essential for the correct refolding. The results also revealed that higher solubility in the presence of higher concentration of L-arginine [>500 mM] or pH [>10] is not associated with higher activity

Amlodipine ameliorates up-regulation of ET-1 in left ventricle of hypercholesterolemia rabbits

In addition to antihypertensive effects, amlodipine may exhibit cardiovascular protective effects in heart tissue. The aim of this study was to evaluate the effects of amlodipine and/or high cholesterol diet on blood, heart tissue concentration and mRNA expression of endothelin-1 [ET-1] in male New Zealand white rabbits. A total of 40 male New Zealand rabbits were divided into four groups: the normal control groups, normal group receiving amlodipine, high-cholesterol diet group and high-cholesterol diet with amlodipine group. After 8 weeks, all the animals anesthetized and blood or tissues samples were collected. After 8 weeks of a high cholesterol diet, the group with such a diet had a significantly higher ratio of left ventricle [LV] weight to body weight than the control group [P = 0.0001]. After treatment with amlodipine for 8 weeks, ET-1 level was reduced considerably in comparison with the control [P= 0.01] and high-cholesterol diet groups [P= 0.01]. Amlodipine consumption caused significant reduction [P=0.01] in the level of ET-1 in heart tissues of high-cholesterol diet group but it had no remarkable effect on the reduction of heart tissue ET-1 in amlodipine group compared with the control group. The present study demonstrates that ventricular prepro-ET-1 mRNA quantitatively increases in the high-cholesterol diet rabbits which results in development of ventricular hypertrophy. It seems that the treatment with amlodipine retards the progression of LV hypertrophy through attenuation of ET-1 levels independent of lipid changes

PER-1-type extended-spectrum beta-lactamase producing acinetobacter baumannii isolated from clinical specimens in Tabriz, Iran

To determine the prevalence of PER-1 gene type ESBLs producing isolates of A. baumannii in clinical specimens. During March 2008 to June 2009, a total of 100 A. baumannii isolates were recovered from clinical specimens of hospitalized patients in Imam Reza hospital in Tabriz. These isolates were subjected to susceptibility tests using five selected cephalosporins according to CLSI guidelines. Screening for ESBL production was carried out by confirmatory tests including DDST and CDM. The prevalence of PER-1 gene in these isolates was also determined by using PCR. All of A. baumannii isolates in this study were resistant at least to one of the selected cephalosporins. Sixty four percent of isolates exhibited a>5 mm zone size enhancement in the CDM test, whereas 53% of them gave positive results by DDST as ESBL producer. Collectively CD method and DDST showed 70% of A. baumannii as ESBLs producer, of which 51% had PER-1 gene. This research shows high prevalence of PER-1 gene type ESBLs producing A. baumannii isolates in Tabriz which is strong evidence for their resistance to cephalosporins

pattern of brain-derived neurotrophic factor gene expression in the hippocampus of diabetic rats

The aim of this study was to evaluate the effects of regular exercise in preventing diabetes complication in the hippocampus of streptozotocin [STZ]-induced diabetic rat. A total of 48 male wistar rats were divided into four groups [control, control exercise, diabetic and diabetic exercise]. Diabetes was induced by injection of single dose of STZ. Exercise was performed for one hr every day, over a period of 8 weeks. The antioxidant enzymes [SOD, GPX, CAT and GR] and oxidant indexes with brain-derived neurotrophic factor [BDNF] protein and its mRNA and apoptosis were measured in hippocampus of rats. A significant decrease in antioxidant enzymes activities and increased malondialdehyde [MDA] level were observed in diabetic rats [P=0.004]. In response to exercise, antioxidant enzymes activities increased [P=0.004]. In contrast, MDA level decreased in diabetic rats [P=0.004]. Induction of diabetes caused an increase of BDNF protein and its mRNA expression. In response to exercise, BDNF protein and its mRNA expression reduced in hippocampus of diabetic rats. Diabetes induced oxidative stress and increased BDNF gene expression. Exercise ameliorated oxidative stress and decreased BDNF gene expression